DESCRIPTION: We have developed a new-generation scFv recombinant immunotoxin (RIT) that holds promise for treatment of melanoma due to its potency and low immunogenicity. Despite its promising in vivo efficacy, it is rapidly cleared from systemic circulation, so that it does not live up to its therapeutic potential suggested by its high affinity for its target and its potent cytotoxicity. In order to unleash its full therapeutic potential, we hypothesize that improvement in the pharmacokinetics and biodistribution of the RIT are necessary. PEGylation-the attachment of poly (ethylene glycol (PEG)- is a powerful method to improve the pharmacokinetics and tumor accumulation of protein drugs. Unfortunately, current methods of PEGylation have poor yield, limited site-selectivity and result in heterogeneous conjugates with compromised bioactivity. We have developed a new method -termed InStealth- that solves these problems by growing a site-specific and stoichiometric (1:1) protein-polymer conjugate with high yield directly from the N- or C-terminus of a protein of interest by atom transfer radical polymerization (ATRP). In previous studies with model proteins, we have shown that a poly(oligoethylene glycol methyl methacrylate) (POEGMA) conjugate has significantly enhanced pharmacokinetics and better tumor accumulation than the parent protein. The overall objective of this proposal is to directly grow a POEGMA conjugate from the N- or C- terminus of the F6V-IT fusion by in situ ATRP to create a site-specific, stoichiometric (1:1) protein-polymer conjugate with long circulation, and improved tumor accumulation. Our Specific Aims are: 1): Generation and characterization of F6V-IT. The F6V-IT recombinant immunotoxin that exhibits extremely low B cell and T cell epitope immunogenicity will be genetically synthesized and it's in vitro activity will be evaluated on different melanoma cell lines. 2): Synthesis and in vitro characterization of F6V-IT POEGMA conjugates. ATRP will be used to synthesize POEGMA conjugates directly from the N- or C-terminus of the F6V-IT with systematically varied POEGMA molecular weight. The targeting and cytotoxic activity of the conjugates on relevant melanoma cell lines will be characterized in vitro. 3): In vivo evaluation of F6V-IT POEGMA conjugates. The in vivo pharmacokinetics, organ bio distribution, and therapeutic efficacy of lead candidates identified in Specific Aim 2 will be investigated in human melanoma xenografts in athymic mice. The outcome of this proposal will be a novel therapeutic strategy for melanoma patients: InStealth(tm) conjugation for tumor targeted delivery of a new immunotoxin with extremely low immunogenicity.